Why your DNA is not like a blueprint

By Rich Feldenberg:

When future historians may look back on the 20th century they may critical of humanities violent tendencies, and rightly so. We drove ourselves to the point of near self-extinction with global warfare and the creation of nuclear weapons. But maybe they will also see a flowering of our more nobel side, as well, with the 20th century ushering up a new understanding and appreciation of nature and ourselves. Many areas of science saw exponential advancements, with general relativity, special relativity, and quantum mechanics, all being born in the last century. As important as these fields have become, the areas of life science have arguably had an even larger impact on society and our everyday lives. It was in the 20th century that we learned that DNA is the molecule of heredity, and the structure of DNA was described by Watson and Crick in 1953 as the now famous double helix. With improvements in the methods of molecular biology in the 1980s and 90s, genomics has lead us to a more complete understanding of the underlying mechanisms of disease and how the normal processes of life operate, develop, and evolve.

The word DNA is now common in the everyday vernacular, even if not everyone remembers that stands for deoxyribonucleic acid. Also nearly everyone has some idea that DNA is vital to genes and inheritance, and is used in forensics, paternity testing, genetic testing for disease mutations, and for mapping phylogenetic trees to understand the relatedness of all life. Somehow, though, we’ve been repeatedly told that DNA is like our blueprint. That it gives the plans for creating you and me, and anything else that has DNA. That analogy is a little misleading, as DNA doesn’t act as a blueprint at all. Looking at the full genetic code of an organism wouldn’t help you know very much about what that organism looked like. The only way you might really infer this from the genetic information would be by comparing the DNA sequences to other organisms that you already know a lot about. If the DNA you are looking at was very close to the DNA coding for octopus and squid then you could guess that this organism looks cephalopod-like. The DNA would not tell you the body plan by analyzing just the code on its own, however.

So how should we think about DNA? Is there something else we can compare it to that would make a more accurate analogy? Well, instead of being a blueprint, like a technical drawing that lays out the structural relationships of each part to the other parts, it is really more like a running computer program. DNA is a lot more like a large collection of computer programs, some are always running, and others are only running at certain times or in certain cell types. The DNA is giving instructions that are carried out by hardware running the code. In this analogy the DNA is the software and the cell and its molecular machinery is the hardware running the software. Software without hardware is hopelessly ineffectual, and hardware without software is nonfunctional. They both need each other to function. The DNA needs a living cell to carry out its instructions. In the proper setting these instructions are powerful, producing a whole human being from just a single cell, as it did with you during your 9 months of gestation in the womb. So how does it work?

Well, it’s important to recall how the information stored in DNA is interpreted by the cell’s internal machinery. The DNA itself is made of two long strands, forming the famous double helix. Each strand is made of sequences of nucleotide bases, and there are four nucleotide bases to choose from in the DNA alphabet – The DNA letters are A, C, G, and T. These letters are chemically distinct nucleotides, and you can picture a gene as being a string of these letters that make a unique sentence. A typical gene may be hundreds to thousands of these letters in length. For example, the human gene for the AVP-2 receptor, found on the X chromosome, codes for a protein located on the cell surface of certain kidney cells, and is critical to regulating normal water balance. The gene contains 4676 of these DNA letters.

Starting from letter one to ten of the AVP-2 receptor DNA code, the letters read out as CTGCCCAGCC, but all 4676 letters of the DNA code for this gene are known and can be found in genetic databanks. Each strand of DNA has a complementary strand where every base in one strand pairs to another base in the other strand. A:T are pairs, and C:G are pairs. In other words, if you know the sequence of one strand you can easily deduce the sequence in the other strand, so for our first ten bases in the AVP-2 receptor – CTGCCCAGCC we know that the complementary strand would have to be GACGGGTCGG, based on the pairing rule. It is this complementary base pairing that makes it possible for DNA to replicate itself. Each strand serves as the template for making a new DNA strand. The double helix just needs to be unwound at the right time, the complementary bases added to each of the now single stranded DNA strands, and you now end up with two identical double helix DNA molecules, where you initially had just one. This has to happen for cells to divide so both of the new cells created from the original single cell has the same DNA as the original.

There are two major cell processes involved for turning the DNA code into protein. For the most part it is the protein that does the actual work in the cell, while it is the DNA that is the code-like programming being run. The first process is transcription, where the DNA code is converted or transcribed into an RNA code. The second process is translation, where the RNA code is converted or translated into the protein product. For the sake of simplicity, we are only talking about protein-coding genes, but there are many non-coding RNA genes, as well – we’ll save that topic for another day.

During transcription a molecular machines known as RNA polymerase interprets the DNA code and converts it into an RNA code, in the form of a single strand of messanger-RNA. RNA is quite similar to DNA except for a few key differences. One distinction is that it is single stranded rather than double stranded like it’s DNA cousin. Another is that it contains an extra chemical group called a hydroxyl that is lacking in DNA, and a third distinction is that whereas the letters in DNA are A, C, G, and T, in RNA the T is missing and a U is there in its place. The RNA alphabet, therefore has the letters A, C, G, and U, with A:U forming pairs and C:G forming pairs. The RNA can then be transported to the cell machinery used to make protein, and the DNA (the original code or source code) can remain safe in the chromosome – only the transcribed copy is sent out.

The messenger-RNA (mRNA), finds it’s way to the ribosomes which are complex molecular machines that take the RNA code and make the actual protein. The RNA code is read by the ribosomes with every three bases forming a codon that specifies an amino acid. The protein is a string of amino acids. A few codons also tell the ribosome where the protein ends, and are called stop codons. The protein may still have a few steps to go before it is fully functional. For example it may need to have certain sugars or other chemical groups added at particular locations. It also needs to be folded into a very specific 3-dimentional shape, and it may need to associate with other proteins to form a part of a larger protein complex. Then it may need to be transported to specific sites in the cell, or even exported out of the cell, to do a job located in a different place in the body.

So why is our DNA not like a blueprint? Well, even if you could read the entire DNA code for all the protein producing genes, you would only see the ingredients that the DNA was coding for. That is far from a blueprint that might show you the structure of a building, where it’s doors, windows, elevators, stairwell, and so on, are located in spacial relation to one another. Knowing the protein products only gives you a list of ingredients. How those ingredients interact together, in time and space, is what creates an organism. The genetic code is a set of instructions that is executed on the code reading machinery of a living cell. The beauty of it is that not all the genes are transcribed at the same time and in the same amounts. Only a fraction of genes would be operational at any given time, and in a complex multicellular organism, only certain genes will ever be transcribed in any particular cell type. That is what makes a kidney cell different from a brain cell different from a cell in the heart muscle, and so on. Every cell has all the genetic programs, but only runs a subset of the total programs necessary for its own type.

DNA without the code reading cell machinery can do nothing on its own, which is why the vital flame of life must be passed down from living cell to living cell, uninterrupted since the very beginning of life itself. The genetic program is sophisticated enough that it causes genes to be transcribed that produce proteins that are themselves transcription factors secreted out of the cell to instruct neighboring cells as to which of their genetic programs to begin running. It is this complex coordination, leading to the switching on or off of particular genes in other cells, that starts the process of building a whole multicellular organism. In this way it is not just the genetic program that is necessary for building a animal, or person, or plant, but the local chemical environment that the program of each cell finds itself living in. The chemical neighborhood is just as important as genetic constituency.

In the language of Object Oriented Computer Programming, like Java for example, we might say that the complete genome of an organism is a program with many Classes (genes), and that when these classes are run instantiate Objects (proteins). Each and every cell in a body has the same program, but depending on it’s interaction with neighboring objects will Call only certain classes for use at any given time, and in some cases will never use particular classes that it has access to. These objects then go on to run all the functions necessary for that cell, including affecting other cells to call on certain objects in some cases. A human kidney cell has the entire “Human Program” as part of its software, but will only call on the classes used by a kidney cell, because it was derived from a cell that at one time could use all classes (Pluripotent stem cell), but at a certain point was instructed by its chemical environments to only allow use of the kidney classes. In other words, it differentiated into a kidney cell, thereby losing the ability to be a different cell type.

This is one reason, that even though we have completely sequenced the human genome, we still have a very incomplete understanding of what most the the genes are doing. Just by looking at their code it is not easy to determine what their affect is in a whole organism. The computer analogy may not be the perfect analogy, but it does illustrate the problem much better than the typical blueprint analogy does.

 

Other interesting things about DNA, and other fun topics:

1. “Intron Retention: a common cause for cancer“.  by Rich Feldenberg. ZME science. 1/25/2016.
2. Alternative Splicing.  Wikipedia.
3. Non-coding RNA.  Wikipedia.
4. “Why the Horta would not have looked like a rock monster“.  Darwin’s Kidneys.  June 18, 2015.

Why the concept of species is more fuzzy than you might think

By Rich Feldenberg:

The term species is viewed as a fundamental unit in biology. We are the species Homo sapiens, and we love to classify ourselves and other creatures into unique categories, giving them qualities that set them apart from other creatures. Charles Darwin’s evolution by natural selection gave the first proofs that all living organisms today descended from a common ancestor, which branded into an ever growing number of different evolutionary paths, resulting in a tree of life.  The trunk being the last common ancestor (LUCA) and all the tiny twigs at the ends representing all species that have ever existed. But is this really an accurate view of the living world – each species of organism occupying its own unique little cubby, completely distinct from it’s fellows in the cubbies next door? We have learned a lot since Darwin’s important discovery of Natural Selection. Modern biology tells us that, while evolution is on firm scientific ground, the concept of the species is less so. We humans have a tendency to think in a discontinuous way – that things do fall into distinct categories – that there is a separate essence that each thing has unto itself. That may be one reason why evolution is a difficult concept to accept for some people, because if each thing has it’s own essence of being, you can’t change it into something else. This idea was demonstrated in an experiment where children were told a story about a witch that turned a frog into a rabbit. The frog now looked like a rabbit, acted like a rabbit, preferred to eat carrots and not flies, wanted to hang out with other rabbits, but when the children were asked if this animal was now a rabbit they said it was really a frog. It’s underlying froggy essence could not be altered by the witches spell.

It turns out that the concept of species is really not a discontinuous one at all. It is a continuous variable, and that may be a difficult idea to wrap your head around. In medicine some diagnoses are continuous and others discontinuous, and in some others it may be difficult to know for sure. For example, having 6 fingers on your hand is discontinuous (you either do or you don’t), but systolic blood pressure is a continuous value, with a range of anywhere from 0 to somewhere quite high like 250 or perhaps rarely 300, with most people being in a certain range, like from 100 to 140. So why is the species also a continuous variable? Isn’t a rabbit a rabbit, and a frog a frog? Certainly a human is a human, and not a chimpanzee, right?

Well, keep in mind that names are just there for our convenience. How close they approximate reality may vary depending on the purpose of the name, and how good we are at understanding what we are describing. Ideally, a name would completely describe reality, but that will never be the case because a name is just a short hand way of talking about something else. Species tells us about taxonomic ranking. This is meant to help us determine which ancestors all the members of the species have in common, and how closely related those members are to members of another species. The problem arises in that the there is no defined line in the ground where one species ends and the next begins. One definition of species asserts that members of one species can not reproduce to have fertile offspring with members of any other species. This definition isn’t technically correct. The reason that this seems true most of the time, is that the many of the ancestral species happen to have died out, so it creates the appearance of very distinct groups of organisms (each in a separate and walled off little cubby).

Every generation is the same species as its parents and the same species as its own offspring. If this is true how could new species arise? It is precisely because the changes that occur due to evolution do so over much longer periods of time than a mere few generations. An organism could reproduce with a member of the prior generation, and if transported back in time to members from tens or hundreds of generations prior. At some point, however, there will be enough structural and/or behavioral differences that reproduction would no longer be possible.

Say that we took an organism back in time (it could be any organism, even a human). When we took him or her back 50,000 generations we find that he/she was able to reproduce with the contemporary population, but couldn’t reproduce with the individuals from 100,000 generations earlier. Now if we go back 50,000 generations from our starting point and take a subject from that era (one that could easily reproduce with our original organism) and then take him or her back to 100,000 generations before our starting point (50,000 generations before this individuals time) we find that it can reproduce with an individual from that long distant time period. So the original could reproduce with a subject from 50,000 generations ago, but not 100,000 generations ago, and the individual from 50,000 generations ago could reproduce with one from 50,000 generations ahead or behind its own time.

Since in the real world, those ancestors are mostly extinct it give the illusion of a discontinuous landscape of species. Richard Dawkins gave an excellent example in his book, “The Ancestors Tale” when he described ‘the salamander’s tale’. In that example Professor Dawkins described several species of salamander that live along a ring of high elevation in Central California. The ring forms a physical geographical structure that makes the species adjacent to any salamander’s location available to them, but prevents the species from interacting with species on distant parts of the ring. At the southern end of the ring are two distinct appearing species Ensatina eschscholtzil and Ensatina klauberi. E. eschscholtzil is brown and lauberi is spotted, and the two species, while in contact with each other do not interbreed – the very definition of separate species! That’s all well and good except that as you go up north on either side of the ring there are more species still, and each of these can reproduce with the species neighboring it.

It’s likely that the ancestral species arrived at some time in the past in the north. Two descendant populations emerged, one going south by the eastern route and the other going south by the western route. If all those other species on the ring had gone extinct we would be left with two species that could not interbreed and there would be nothing very special about the story. Those other species did not die out, however, and there is a continuous ring of salamanders that can reproduce with others of similar species, except in the case of the two southern species. As Dawkins says in the book, “Strikes a blow against the discontinuous mind”. The situation for these salamanders reveals what the situation would be like for any species (human included) if all of our ancestors were alive today. There would be a continuous stream of organisms that can interbreed with those close to them in time, but only over longer time scales do differences add up that make them distinct enough that interbreeding is no longer possible.  The sides of the ring represent a common ancestor, diverging and over time becoming two separate species, but having a path of “able to interbreed” individuals all the way down.  

In another post, I’ll illustrate another problem with the “tree of life” concept.  In actuality the tree concept is complicated by “lateral gene transfer” – basically genes being swapped by other organisms of different types.  This is very common in bacteria, but also seems to happen to some extent in more sophisticated organisms.  In any case, the idea of species should be used as a useful placeholder, but has important limitations.

Reference:

1. Species, Wikipedia.
2. “The Ancestor’s Tale: A Pilgrimage to the Dawn of Evolution”, Richard Dawkins. 2004.

3. Another clever Mesign by Mother Nature.  Darwin’s Kidneys.  July 8, 2015.

 

Happy Darwin Day 2016

February 12th, 2016 is Charles Darwin’s 207th birthday. Charles also happened to share the exact same date of birth with Abraham Lincoln – so happy birthday Mr. Lincoln, as well! Since this blog is dedicated to science, with a special emphasis on evolution, and in fact, has the name Darwin in the title, I want to be sure to honor our dear Mr. Darwin properly.

There are Darwin Day celebrations planned in the USA and around the world, but no ‘Official Darwin Day’ is recognized nationally. That could change as some efforts are being made to make it official. In fact, this year the Governor of Delaware declared an official Darwin Day in his state. In some cities there are lectures or parties to celebrate.  The Center for Inquiry has a take action page, where you can send your name in a letter  to members of congress to express the importance of creating a Darwin’s Day for public education of science.

Charles Darwin’s theory of evolution was the beginning of modern biological science. As the Russian evolutionary biologist Theodosius Dobzhansky is quoted as saying, “Nothing in biology makes sense except in the light of evolution”. Evolution is the thread that binds all of biology together. Every aspect of biology, from molecular genetics, embryology, comparative anatomy, populations and ecosystems all “make sense in the light of evolution”.

Darwin’s theory was a realization of origin from common decent. Evolution does not address the emergence of life from non-biological origins, but does an excellent job explaining the illusion of design seen in the complex structures of the living world. Of course, Darwin realized that the illusion was the product of natural selection working on variations in living things. Darwin had no idea about genetics, DNA, mutations, and so on, but as those fields of biology developed they only reinforced Darwin’s big idea. It could easily have been otherwise. If evolution by natural selection was not how the world worked, then molecular genetics, phylogenetic, developmental biology, and so on would not have provided additional support to a 150 year old theory. Yet, all these modern sciences fit in perfectly, continuing to build on the original theory. Even without the fossil record modern biology would still point the way to evolution. By the way, the fossil record also supports evolution, and has only become more robust during the last 150 years as many more fossil species have now been discovered.

I’m sure Darwin would have been delighted to learn about genes, how new mutations arise by damage due to radiation, chemical mutagens, or simply errors in the normal process of DNA synthesis. He would have loved to see how the genome is cluttered with the remains of dead viruses, pseudogenes, copying errors that we have been copying and passing down to our children for geological eons. And he would have certainly understood that we can see our degree of relatedness to any living species on the planet by looking at, not just the working genes and how closely they match to us, but also these dead viruses and pseudogenes.

Darwin’s voyage on the H.M.S Beagle remains one of the most exciting and most epic expeditions of discovery in history – certainly one of the most productive, since it resulted in much of the data Darwin needed to formulate his theory over the next several decades. Darwin was an amazing naturalist and keen observer. There is hardly any area of natural science of his time that he didn’t seem to make some meaningful contributions. Not just in biology but in geology, as well.

So this Darwin’s day I plan to celebrate at home with my family. Perhaps have a piece of Common Decent Cake or Evolution Pie, learn something new I didn’t know about evolution, and honor our Dear Mr. Darwin.  Let me know how you plan to celebrate.

Other Reading:

1. Darwin Day:  Wikipedia
2. Natural Selection:  Wikipedia
3. Youtube.  Climbing Mount Improbable.  Lecture by Richard Dawkins.
4. OxoG is how radiation turns your own water against you.  Darwin’s Kidneys blog
5. Cytosine Deamination.  Darwin’s Kidneys blog.  (another mechanism of mutation).
6. Another Clever Mesign by Mother Nature.  Darwin’s Kidneys blog.  New word mesign to differentiate apparent design in nature from when we mean a designed object.
7. How our ancestors promiscuous genes became more discriminating.  ZME Science. Feb. 9, 2016.  Article on how gene families arise by gene duplications.

Gene Drives: so you want to change the world!

By Rich Feldenberg:
Want to change the genetic landscape of whole populations and ecosystems? Tired of having to do it the old fashioned way by genetically engineering one organism at a time? Well now there’s Gene Drive! The fast and efficient way to spread your desired genetic design! Just send $19.99 plus shipping and handling for your Gene Driver Kit today!

If the “Fake Advertisement” in the paragraph above, made it sound as though the Gene Drive concept is some crazy kind of internet scam that is to good to be true, actually nothing could be further from the truth. Well no, you can’t just send in money for a gene drive kit yet, but it turns out that gene drives are real, they’re awesome, they’re controversial, and they can in principle, change the gene pool of an entire population of an organism. In fact, this method of gene editing is so new that very few experiments have even been done, and its founder, Kevin Esvelt, feels that the technology is so powerful that he wants to put a halt on experimentation until society can come together and discuss whether we collectively feel this is an area of science we should pursue, not just one that we can pursue. To understand gene drives we first have to remind ourselves of how the CRISPR-Cas9 system works, which I reviewed in an earlier Darwin’s Kidney post.

Briefly, the CRISPR-Cas9 system is a new and powerful gene editing technique that can be used in living organisms. This system is found naturally in many bacteria, as part of their immune defense mechanism against viral attack. There are two major parts to the system. The first is a guide RNA and the second is the Cas9 enzyme. The guide RNA is a small strand of RNA (somewhere around 20-40 base pairs in length). When the guide RNA finds a perfect base pair match with a DNA strand somewhere in the cell, the Cas9 enzyme cuts that piece of DNA. In the case of bacteria, this allows the them to match one of their guide RNAs to a sequence of DNA from an invading virus, then cut the viral DNA, which disables the virus from taking over the bacterial cell. The guide RNA came from a previous viral attack that the bacteria survived, and when the bacterial enzymes chopped up the invaders DNA into small bits, some was incorporated into CRISPR so that exposure to that same virus the next time would quickly result in recognition by the bacteria – an immune system! In the last few years scientists have discovered how to make guide RNA for any desired gene, and along with the Cas9 enzyme, can then “snip out” the gene or any piece of DNA in question. This can be used to silence genes, or can also be used to replace genes if the cell has access to a DNA sequence that can fill the gap left by the Cas9 enzyme. This may turn out to be a great way to cure genetic diseases through gene therapy.

Gene drive systems, take this concept a step further. Gene drives rely on the gene editing to take place in germ line cells versus somatic cells. Germ-line cells are the cells that will become egg or sperm, and will be used to create new organisms through sexual reproduction. Somatic cells are all the other body cells, such as skin, kidney, brain, pancreas, etc. If a gene is edited in a somatic cell, that change will effect the organism in whom the change was made, but would not be passed down to the next generation.

As an example, lets say you want to be able to provide gene therapy for a genetic disease such as Nephrogenic Diabetes Insipidus (NDI). This disorder is X-linked, meaning that the gene is on the X chromosme. Since males have an X and Y chromosome, with the X coming from their mother and the Y coming from their father, if the mother’s X chromosome has the mutant gene for NDI they will have inherited the disease, which leads to the kidneys inability to regulate water loss. People with this disorder can die of dehydration because even when dehydrated they continue to produce too much urine. A female, having two X chromosomes, one from her mother and the other from her father, might be a carrier for NDI if her mother’s X had the mutant NDI gene, but she still wouldn’t develop the actual disease since her normal NDI gene from her father’s X chromosome will compensate.

In principle you could use the CRISPR system to edit the defect gene, so that the male patient with NDI can now regulate water loss through the kidneys normally. There is still no way to really do this yet. You would need to deliver the CRISPR-Cas9 system, to the appropriate kidney cells of the affected individual. At the present time, a way to target and deliver the system is still not available, but if it could be delivered to the kidney cells it would excise the defective DNA. The cells own repair mechanisms will then look for a replacement to fix the DNA break made by the CRISPR-Cas9. If the normal gene was also delivered to the cell it will be incorporated into the place where CRISPR-Cas9 made the break. This will result in having removed the defective disease causing gene and replaced it with the normal healthy gene, and should therefore cure the disease – Nephrogenic Diabetes Insipidus kidney disease in our example. However, even if this could really work – its never been tried yet for this disease – but was unsuccessfully attempted for Hemophilia, the cured individual would still be able to pass the disease on to their children. The reason is that only the kidney cells were altered, and not the germ-line cells.

Gene drives, on the other hand, effects the germ-line, but they have an even bigger, more ingenious twist to their potential to alter future generations. With gene drives, in addition to supplying the new gene, the genetic code for more CRISPR-Cas9 is also inserted into the target genome. So here is how it might work. Let simplify the example by calling the two alleles of the gene (one allele comes from mom and the other from dad) as Normal and Engineered. It could be any gene in the genome that you’re interested in, such as the gene for making insulin or for making neurotransmitters in the brain, or transcription factors that tell more genes what to do. In this example we want the Engineered gene to take over because it has some trait we have engineered for it that we find desirable. It could be to fix a defective gene or it could be to give the organism some new property. We’ll get to some examples of new properties shortly.

 

The first step might need to take place in the lab when the organism at its earliest stage, the fertilized egg. You place the CRISPR-Cas9 into the fertilized egg with guide RNA that recognizes the Normal gene. You also place the Engineered gene in the cell to replace the Normal gene once Cas9 has cut it. The cell will now have the Engineered gene as part of its entire genome. This will effect both somatic cells and germ-line cells since the fertilized egg will continue its job of dividing into more and more cells, which will eventually become all the cells of the body. Eventually this organism will develop into an adult and find a mate to produce more offspring. The offspring will have an approximately 50% chance that the Engineered gene will be passed on to the the next generation. That is because each offspring will get one copy of Engineered gene from our genetically modified organism and the other gene from its mate, which would carry the Normal gene version since it was never modified. So this is where the special ingenious twist comes in!

Not only does the gene you inserted into the fertilized egg contain the DNA of your engineered gene, but it contains the DNA for making a CRISPR-Cas9 system, as well. This CRISPR-Cas9 is hidden somewhere in the middle of your Engineered gene so that the cells DNA repair enzymes don’t recognize it as being novel to the cell. They only recognize the ends that need to fit in the space that Cas9 cut out. So in this way, the gene we pasted into the genome is Engineered-CRISPR-Cas9. Now when the cell transcribes that gene the CRISPR-Cas9 is also transcribed which leads to a guide RNA and a working Cas9 enzyme. The guide RNA will then match to the Normal gene and Cas9 will cut it. This is important because when the genetically modified organism mates with a wild type organism the offspring will have one Normal gene from the wild type and one Engineered-CRISPR-Cas9 gene from the genetically modified organism. CRISPR-Cas9 then gets transcribed, seeks out the Normal gene, and replaces it with the Engineered-CRISPR-Cas9 gene, so the offspring actually ends up with two copies of the Engineered-CRISPR-Cas9 gene. In this way the rate of transfer of the Engineered gene, to successive generations, goes from 50% to 100%. The Engineered-CRISPR-Cas9 gene effectively edits every other allele that matches its guide RNA, turning it into the Engineered gene. Now the gene can spread rapidly through a population because the odds are always in favor of this gene being passed down to all offspring. See the excellent Mosquito chart in the following article I’ve linked to in order to get a visual on how the inheritance would be effected.

It should be pointed out that this is only effective for organism that reproduce sexually. Asexually reproducing organisms (such as bacteria) won’t be influenced by this mechanism. For organism that have short generation time this is ideal. One proposed problem that gene drives might be able to solve would be in the fight against malaria. Malaria kills millions of people each year (see Darwin’s Kidneys article: Diseases with an Upside). If mosquitos were released into the wild, that were engineered to have a malaria resistant gene and also the CRISPR-Cas9 system, then that gene would spread rapidly throughout the mosquito population. The result would be malaria resistant mosquitos and possibly an end to suffering and death in many parts of the world due to this parasitic infection.

There’s no guarantee, however, that the malaria organism – Plasmodium – would not find a way to evolve around the mosquito’s malaria resistance given enough time. There is also no guarantee that the malaria resistant gene might not somehow decrease the “genetic fitness” of the mosquito making them less likely to survive and reproduce. Mosquitos would be an ideal organism for this type of engineering, however, since they have a rapid generation time, so within several years to decades a gene system of this type could theoretically pass to all members of the population. Humans, on the other hand, reproduce slowly so a gene drive in humans would probably take hundreds of years to spread through the population. Still, you could imagine an attempt to eliminate many genetic diseases completely from existence by using gene drives that over the course of centuries might be effective. One could also imagine the ability to produce a civilization of future generations of humans that are more intelligent, more rational, less violent, more empathetic, and so on, if the genes involved in producing those traits could be identified. It is harder to imagine, however, that society as a whole would ever agree to such a mass alteration of the human genome – creating something beyond human – by directing human evolution in a desired direction. Its too early to know if such changes to the human genome could even be done safely without creating damaging consequence that are impossible to predict. I’m not necessarily advocating for changing the human race for the better, but more just advocating for discussion of the potential positive and negative effects might result from such grandiose dreams.

Because the implications for gene drives are so powerful and large scale, there is currently a call for a hold on research until the ethical considerations can be more fully considered. I think this seems wise at our current state of understanding. Changing an ecosystem could have unforeseen consequences. There may be ways to alter some behavior in organisms with gene drives that would not necessarily eliminate those organisms from the ecosystem – and so may have a mild impact on the ecosystem as a whole. For example, one could engineer a pest to dislike the taste of a crop that it normally damages, and therefore protect the crop without the need for as much pesticide use. The pest is now no longer a pest, but remains in the ecosystem where it can feed on other plants and remain part of the normal food chain for other organisms. Could gene drives be used to engineer plants to more efficiently remove CO2 from the atmosphere, and combat global warming while increasing crop yields?

Gene drives are an exciting new method of changing the genetic makeup of populations of organisms. Whether they will be used to prevent diseases like malaria from killing so many or making crops less prone towards pests and therefore reducing the amount of insecticides released into the environment, is up to society at large to decide if we are ready to pursue such far reaching technology. My hope is that we may find ways to safely use gene drives to improve life on planet earth for ourselves and our fellow species.

References:
1. “Genetically Engineering Almost Anything” by Tim De Chant and Eleanor Nelson, Nova Next. July 17, 2014.
http://www.pbs.org/wgbh/nova/next/evolution/crispr-gene-drives/
2. “Gene Drives and CRISPR could revolutionize ecosystem management”, by Kevin Esvelt, George Church, and Jeantine Lunshof; Scientific American Blog. July 17, 2014.
http://blogs.scientificamerican.com/guest-blog/gene-drives-and-crispr-could-revolutionize-ecosystem-management/
3. Gene Drive Wikipedia: https://en.wikipedia.org/wiki/Gene_drive
4. “Gene editing in Humans”; Neurologica blog by Steven Novella; Nov. 19, 2015
http://theness.com/neurologicablog/index.php/gene-editing-humans/
5. “CRISPR: what’s the big deal?”, Darwin’s Kidney blog by Rich Feldenberg. Nov. 28, 2015.
http://darwinskidneys-science.com/2015/11/28/crispr-whats-the-big-deal/
6. “Can we genetically engineer Rubisco to feed the world?”; Darwin’s Kidney blog by Rich Feldenberg.
July 22, 2015.
http://darwinskidneys-science.com/2015/11/28/crispr-whats-the-big-deal/
7. “Diseases with an upside”; Darwin’s Kidney blog by Rich Feldenberg. July 29, 2015.
http://darwinskidneys-science.com/2015/07/29/diseases-with-an-upside/
8. “Live at the NESS: New Dilemmas in Bioethics”; The Rationally Speaking Podcast. April 24, 2011.
With Massimo Pigliucci and Julia Galef as hosts.
http://rationallyspeakingpodcast.org/show/rs33-live-at-necss-new-dilemmas-in-bioethics.html

9. “Sculpting Evolution”; website of Kevin Esvelt, PhD.  Founder of gene drives.   http://www.sculptingevolution.org/kevin-m-esvelt

 

 

 

Diseases with an upside!

Diseases with an upside.

By Rich Feldenberg

Since life’s earliest emergence on planet earth, disease has been our constant and unwelcome companion.  Even the first single celled organisms were susceptible to break down, nutritional deficiencies, and harmful genetic mutation.  When single celled life upgraded to the multicellular stage, finally becoming large, it was then susceptible to a host of new disorders, such as cancer that interfered with the organization and growth of cells that now had to survive as part of a collective.  Humankind is no different than the rest of the animal kingdom in this regard, and throughout human existence disease has lead to untold suffering, death, and at times the threat of total extinction.  It may therefore be surprising to learn that some diseases confer protection against other types of illness, and this seems to account for the high prevalence of some of these disorders in the human population.  If the protective benefit of the disease mutation on a large portion of the population outweighs the suffering and death of a small portion of the population, natural selection will swing the balance in favor of keeping those mutations in the gene pool.  Not only may the disease mutation simply persist in the gene pool, but it may become very prevalent because it is selected for in the right environment, where the other illness it protects against is a major threat.   To illustrate how this works I’ll give some detail on two well known examples of diseases and their upside – in other words, how they protect against other threats to our species.

The first example is that Sickle Cell Anemia (SCA), which has the best documented evidence as to its evolutionary risk versus benefit ratio in its effected population.  Sickle Cell Anemia is a genetic disease that causes anemia (low red blood cell counts), and can lead to painful, and potentially deadly pain crises.  It is inherited as an autosomal recessive trait – meaning that if you receive one copy of the mutated gene from each of your parents, then you have two abnormal copies of the gene (are homozygous, in the language of genetics) and will have the disease.  Each of your parents, however, has only one mutant copy and also one normal copy (is heterozygous), and so is only a carrier (has sickle cell trait) and will not show symptoms of the disease under normal circumstances.

SCA is due to a single base switch in the DNA that codes for the beta-chain of the hemoglobin molecule.  Adult hemoglobin is made of two alpha chains and two beta chains.  This is the major oxygen carrying protein in the blood, although, there are other versions of hemoglobin that are produced (one example is fetal hemoglobin with two alpha chains and two gamma chains).    In SCA, there is a substitution of the amino acid glutamic acid for valine at the 6th amino acid in the beta chain.  Since valine is more hydrophobic than glutamic acid this has the unfortunate consequence of causing the hemoglobin molecules to polymerized and compact together, deforming the shape of the red blood cells (RBCs) that carry them, into a sickle shape – hence the name Sickle Cell Anemia.  The polymerization event is more likely to happen if the affected individual is dehydrated, in a low oxygen state (hypoxic), or otherwise ill with another illness.  The deformed red blood cells can not get through the tiny capillaries very well, causing blockages that deprive tissues of blood and oxygen.   The result is pain and organ damage.

Over time, people with SCA damage their spleen so badly that they lose the its important immune function, which normally you against encapsulated bacterial infections.  These are certain bacteria that are surrounded by a polysaccaride capsule, that helps them to escape detection by the immune system.  Someone without a functioning spleen can then die of these types of infections, whereas those with normal spleens would be able to fight off the infection easily.  The blockages to blood flow due the abnormal sickle shaped RBCs can lead to strokes and to Acute Chest Syndrome.  If people with SCA become infected with the common virus Parvovirus B19, they can develop severe life threatening anemia, with hemoglobin levels that get so low they can develop heart failure.

Sickle cell anemia is common in sub-Saharan Africa, and about 300,000 are born with disease each year.  All the complications of SCA listed above can be fatal so why would this disorder have such a high prevalence?  The answer seems to be that although people with full blown Sickle Cell Anemia are at a most definite disadvantage from a survival aspect, those who are carriers of SCA are protected against another common killer – Malaria.  Malaria is an infectious disease caused by the protozoan Plasmodium.  It has a complex life cycle, part of which is spent inside the mosquito Anopheles, and part is spent inside a vertebrate host – such as a human.  When an infected female mosquito bites a human, the organism is transmitted into the persons blood stream where it travels to the liver, infects liver cells, reproduces, and then is released back to the bloodstream where it infects RBCs.  The symptoms of Malaria include fever, vomiting, joint and muscle pain, headache, and in some cases seizures.  As the Plasmodium organism goes through its life-cycle within the host, from liver to RBC and back again (these are known as the liver phase and the erythrocytic phase respectively), the symptoms return in a cyclical fashion.  In some cases the organism passes through the blood-brain barrier leading to Cerebral Malaria, which is a very serious complication.  Malaria has a high mortality rate if untreated – as would have been the case before the age of modern medicine.

It was observed, early on, that in regions endemic to malaria, people who were carriers of the sickle cell mutation showed resistance to the malaria infection, and that full blown SCA has a high prevalence in those same regions where malaria is endemic.  Further studies confirmed that those individuals who are carries for the sickle cell mutation, do in fact, enjoy a protection due to their gene mutation.  Unfortunately, those with actual sickle cell anemia (homozygous for the gene mutation) are not protected against malaria.  Not only do they have to suffer the fate of SCA, but if they get malaria they have a worse prognosis because the malaria damages their already vulnerable RBCs.

For a long time it was thought that sickle cell trait most likely confers its malarial protection by making it difficult for Plasmodium organisms to infect the abnormally shaped RBCs, and that the abnormal RBCs are removed more readily by circulating macrophages, helping to rid Plasmodium infected cells more readily.  More recent research seems to suggest that the protective mechanism is more complex that that, and involves the up regulation of an enzyme called heme oxygenase-1(HO-1).    HO-1 causes the breakdown of heme, and the release of carbon monoxide (CO), iron, and biliverdin, resulting in an anti-inflammatory effect.  HO-1 is upregulated or produced to a greater extent in RBCs that have the abnormal hemoglobin associated with SCA, and it is the production of CO that seems to have a detrimental effect for the Plasmodium organisms.  It confers protection against cerebral malaria, and decreased mortality for those with sickle cell trait who become infected with malaria.  This might also be the answer to why several other diseases or disease traits have also been observed to offer protection against malaria, such as thalassemia trait and Glucose-6-Phosphate Deficiency.  These disorders might also increase the activity of HO-1.

We’ll move now to another deadly disease that seems to have remained in the population because it offered a survival advantage.  This is the kidney disease called Focal Segmental Glomulosclerosis (a real mouthful) or just plain old FSGS for short.  FSGS can be caused by chronic infections, such as hepatitis or HIV, but many cases are due to a genetic mutation.  It is a subset of the genetic form that may have been selected for to protect against Sleeping Sickness.  In FSGS the tiny filters in the kidneys, called glomeruli, become scarred until they can no longer filter.  This can eventually progress to kidney failure and the need for dialysis or kidney transplant.  Kidney failure is fatal without modern medical care and FSGS is one of the more common causes for young people to be on dialysis.  Its also, often more common and resistant to therapy in African Americans and other people of African descent.

Some people with the genetic form of FSGS have a mutation in a gene called APOL1, and if you are an individual with two mutated copies of the APOL1 gene, your risk of developing FSGS and kidney failure is 17 times higher than if you have two normal copies of the gene.  That adds up to around a 4% chance of developing FSGS over your lifetime if you are homozygous for mutant APOL1.  This mutation is also thought to explain 18% of all cases of FSGS that currently exist.  There are two types of mutations in the APOL1 gene that can increase risk for FSGS kidney disease.  These is the G1 variant, which contains two amino acid substitutions – one is a replacement of glycine for serine at amino acid 342 in the protein (S342G), and the other switch is a replacement of methionine for isoleucine at amino acid 384 in the protein (I384M).  You have to have both of these switches you have the G1 variant.  The other variant is the G2 variant where 6 base pairs are deleted in the DNA coding for APOL1 starting at base 388.  People can have either a G1 variant or a G2 variant, but never have both types.

APOL1 is a protein that circulates in the blood and is part of the high-density lipoprotein (HDL – otherwise known as the “good” cholesterol).  Exactly how the mutated form of APOL1 causes kidney disease is still not known.  What is known, however, is that those individuals with either a G1 or G2 specific gene mutation in APOL1 have protection against African Sleeping Sickness, caused by the protozoan Trypanosoma brucei.  This tiny single celled eukaryotic organism is transmitted to its human host by the bite of the tsetse fly.  It is a common and dangerous disease in sub-Saharan Africa.  In 1990 it caused 34,000 deaths, but the death rate dropped to 9000 in 2010, thanks to efforts of the World Health Organization to prevent and treat the infection.

Those affected by the parasite experience two distinct stages of infection.  In the first stage the victim develops headaches, fever, and severe itching.  This resolves only to eventually progress to the second stage of the disease which effects the central nervous system causing confusion, paralysis, neuromuscular weakness, and sometimes psychiatric illness.  There is a reversal of the sleep-wake cycle, giving the disorder its common name.  Infected persons often sleep in the day and remain awake at night.  Without treatment the disease always ends in the death of its victim.   It can be treated with the drug pentamidine, when in the first stage, or drugs such as eflornithine or melarsoprol for second stage disease.

Like the association of Sickle Cell Anemia and malaria, those geographic regions with a high incidence of sleeping sickness also have a high incidence in the population of APOL1 G1 or G2 variants.  This is because those gene variants protect against the ravages of the Trypanosomes.  The APOL1 variants cause the lysis (breaking apart of the cell membrane) of Trypanosomes that cause sleeping sickness.  The normal gene for APOL1 gives us resistance to other species of Trypanosomes that do infect other mammals, but are unable to harm us.  The sleeping sickness Trypanosome (Trypanosome brucei rhodesiense) is immune to the normal APOL1 since it has evolved a serum resistance-associated protein (SRA) that blocks a portion of the APOL1 protein, neutralizing its anti-trypanosomal action.  Not so for the APOL1 variants G1 or G2, however.  They are able to get around this SRA and destroy the parasite.  From an evolutionary point of view, the advantage of being more resistant to sleeping sickness in an area of high risk, outweighs the cost of having a higher than average chance of kidney disease.  There is no advantage, however, to having these variants if your ancestors originated where sleeping sickness is not a problem, so other populations aren’t found to have these gene mutations.

The two examples of Sickle Cell anemia and Focal Segmental Glomerulosclerosis (APOL1 mutation) are not the only situations where a disease mutation protect us against another illness.   I’ll just briefly mention two more.  Tay-Sachs disease, which is a lethal neurodegenerative disorder in the homozygous state, seems to protect against Tuberculosis in carriers (heterozygotes).   Also Cystic Fibrosis (CF) which usually leads to severe and chronic lung disease in the homozygous state, may have protected against the effects of cholera in the heterozygous carriers.  The CF mutation inactivates a chloride channel called CFTR, in the cell membrane.  Being a carrier for this mutation may have prevented the lethal dysentery that would have accompanied infectious cholera, by preventing water loss in the intestines due to poorly working chloride channels.  It is a very common gene mutation, with 1 in 25 people of European descent being a carrier for the CF gene mutation.

When we think disease we think of the suffering of its victims and the cost to society.  We are often unaware of the balance of the many forces involved, which influence why a particular disease may be so common in a given population.  The factors involved are typically much more complex than we appreciate, and most of them are still unknown to us.  Natural selection is working behind the scenes in ways that are difficult to detect on just a casual examination.  It may be of no consolation to the sufferers of a serious disease, or the family members devastated by a loved ones sickness and loss, but natural selection, with its cold blind eye to pain or suffering, seems to have fixed some of this in place to allow more genes to be passed onto future generations.  Evolution is not directed toward any particular goal and has no empathy or sense of compassion.  It only selects those traits that happen to give the organism the best chance to pass on its genes in its evolved environment.  This is where the human mind comes into play.  Now that we are finally learning to understand the root causes of disease at the genetic and molecular level, we can work to treat, cure, and eradicate disease.  Although we are not there yet, in theory it should be possible to cure a condition like sickle cell anemia with gene therapy.  At the same time, we shouldn’t have to worry about worsening the burden of malaria if SCA were eliminated, since we can also work on better therapies to treat the malaria, and more effective strategies to prevent infection with Plasmodium.

References and other reading:

 

1. “Mystery solved: How sickle hemoglobin protects against malaria”, ScienceDaily; April 29, 2011

http://www.sciencedaily.com/releases/2011/04/110428123931.htm

2. “Sickle Cell Anaemia and Malaria”, Lucio Luzzatoo, Mediterranean Journal of Hematology and Infectious Disease; Oct. 3, 2012.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499995/

3. Sickle Cell disease;  Wikipedia.

https://en.wikipedia.org/wiki/Sickle-cell_disease

4. Malaria;  Wikipedia.

https://en.wikipedia.org/wiki/Malaria

5. Heme Oxygenase-1;  Wikipedia.

https://en.wikipedia.org/wiki/HMOX1

6. “APOL1 Genetic Variants in Focal Segmental Glomerulosclerosis and HIV-Associated Nephropathy”,  Jeffrey B. Kopp, et al., Journal of the American Society of Nephrology;  Nov. 2011.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3231787/

7. “Association of Trypanolytic ApoL1 Variants with Kidney Disease in African-Americans”,  Giulio Genovese, et al., Science, August 13, 2010.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2980843/

8. “A co-evolutionary arms race: trypanosomes shaping the human genome, humans shaping the trypanosome genome”, Paul Capewell, et al., Parasitology, June 26, 2014.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4413828/

9. “A risk allele for focal segmental glomerulosclerosis in African Americans is located within a region containing APOL1 and MYH9”, Giulio Genovese, et al., Kidney International, Oct. 2010.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001190/

10. African Trypanosomiasis;  Wikipedia.

https://en.wikipedia.org/wiki/African_trypanosomiasis

Can we genetically engineer Rubisco to feed the world?

Today’s atmosphere is brought to you by Rubisco.

Fine makers of oxygen since 3.5 billion B.C.

By Rich Feldenberg

If you happen to peak outside on a nice sunny summers day to admire the green grass, shady trees, and pleasant bushes, your field of view is, in actuality, filled with Rubisco, busily helping the plants do their special thing of making sugar and churning out oxygen.  Rubisco is by far the most abundant enzyme on the earth and accounts for 30%, or more of the protein found in the green leaves of plants.  Without it there would be no oxygen producing photosynthesis, so if you’re a fan of breathing then you’re probably going to be happy to learn about Rubisco.  And, If you’re thinking to yourself, “if there is that much of it in the world it must be doing something important”, congratulations, you’d be right!

There are three things I’d like to point out about Rubisco. One is that Rubisco is freaking amazing.  It is an awesome protein with interesting molecular properties, catalyzing fascinating chemistry, and dating back to some of the earliest life on earth.  The second thing is that as amazing as Rubisco is, it is incredibly poorly mesigned (mesigned is my word meaning designed by natural selection).  Rubisco is horribly inefficient and slow, and it’s amazing it hasn’t been fired from it’s post and replaced with a new, younger, more hip version.  And finally, Rubisco could, in principle, be engineered to be much better, possibly increasing crop yields to feed an increasing global population, and removing CO₂ from the air to combat global warming.  Let’s tackle each of these points.

Point One:, Rubisco is amazing.  Without it life on earth would likely still be living as simple single celled mats of slime on the ocean floor in a oxygen free world.  Rubisco is the abbreviated form for the formal name of the enzyme Rubulose-1,5-bisphosphate carboxylase/oxygenase.  Yeah, that’s why Rubisco (rhymes with San Francisco) rolls off the tongue so much nicer, and is way easier to say three times fast.  This enzyme goes way back to the good old days when singled cell organisms ruled the world, and appears to have a common origin in all three of the major kingdoms of living things -bacteria, archaea, and eukaryotes- indicating a very early origin sometime around the time of the Last Universal Common Ancestor (LUCA).  It appears to have arisen even before the evolution of oxygen producing photosynthesis.  There are, by the way, other types of photosynthesis that do not produce oxygen as a byproduct – so called anaerobic photosynthesis- which are less efficient than the oxygen producing types.  Based on the study of related proteins, known as Rubisco-like proteins (RLPs), Rubisco may have evolved from RLPs that performed other enzymatic functions before it was eventually modified to it’s modern role in photosynthesis.

Rubisco is a big protein, that is itself composed of two main types of subunits – the large subunit (L) and the small subunit (S).  There are three major forms of Rubisco, but in most plants and algae, the Rubisco is composed of a combination of eight L-subunits and eight S-subunits.  Rubisco catalyzes the first step in the photosynthetic process taking CO₂ and making it react with the compound ribulose 1,5-bisphosphate (RuBP).  That is way the Rubisco enzyme is named Rubulose-1,5-bisphosphate carboxylase/oxygenase.  In one of it’s reactions, it is carboxylating the substrate RuBP.  This leads to the formation of two molecules of phosphoglyceric acid (PGA), that then go through a metabolic pathway called the Calvin cycle.  The PGA products eventually go on to other metabolic pathways, and the result is sweet sweet sugar!

Space filling model of the Rubisco protein structure 

The L subunit has the active site with a critical lysine residue for binding CO₂.  It actually takes two CO₂ molecules to get things going.  The first CO₂ molecule is used just as an activator for the enzyme’s active site, but isn’t used in the carboxylation reaction.  The second CO₂ molecule is what is used to react with RuBP, and it is this carbon that is added onto the molecule.  The O₂ that is eventually released at the end of photosynthesis does not come from the CO₂ but is taken from water, which is also necessary in the reaction.   The genes for the L subunit of Rubisco are found in the chloroplasts, tiny organelles within the cells, where Rubisco is conducting its important job.  The S subunit is more of a stabilizing part of the protein and its gene is located in the nucleus, and once the protein is made, needs to be shuttled into the chloroplasts.

 

 

Also necessary for the enzyme to function is the presence of an ion of Mg⁺² ,which acts to stabilize the activation site.  This process allows one CO₂ molecule, along with a molecule of H₂O to become incorporated in RuBP.   There is a whole lot of Rubisco in the green leaves of plant to carry out this important chemical reaction.   As we said above, about 30% or more of the protein in the leaves of plants is in the form of Rubisco, so it therefore accounts for a huge amount of nitrogen stored in the biosphere, since proteins contain nitrogen as part of their structure.  

Point Two: Rubisco is so mind numbingly inefficient I am almost embarrassed for our plant cousins.  It turns out that the carboxylase function of Rubisco (you know the really important thing it does by taking CO₂ from the air and attaching it to RuBP to begin the process of making carbohydrate) is not the only reaction it performs.  In fact, it’s very name -the unabbreviated one that is- tells you right off that it it also is an oxygenase.  That is the carboxylase/oxygenase last portion of the name.  This means that Rubisco is not terribly selective for CO₂, but can also react at the activation site with a molecule of molecular oxygen (O₂), which has some chemically similar properties.  This leads to a horribly counterproductive metabolic pathway called photorespiration.  In other word, it is not very selective, and is so nearsighted that it may grab onto an O₂ as easily as a CO₂.  Normally about 25% of the reactions that Rubisco is catalyzing are with oxygen going down the photorespiration pathway.  Also, keep in mind that in the atmosphere today, and for at least the last billion years, the concentration of O₂ has been way in excess of that of CO₂.  The atmosphere these days is 21% O₂ and only 0.04% CO₂, so that makes it even more difficult for poor little Rubisco to discriminate effectively.

Ribulose 1,5-bisphosphate (RuBP)

Photorespiration leads to RuBP being converted into one molecule of PGA and one molecule of 2-phosphoglycolate.  This doesn’t lead to carbohydrate production.  Even worse this uses energy in the form of ATP and released CO₂ into the air.  Totally wrong if you want to store energy from the sun in the form of yummy sugar molecules.  So we can clearly say that Rubisco has a poor affinity for CO₂.  An enzyme’s affinity for its substrate is measured by a characteristic called K_m, and Rubisco’s K_m is kind of wimpy.   This relative non-selectivity may be a reflection of the world in which Rubisco first evolved.  At that time the concentration of CO₂ in the atmosphere would have been much higher and the concentration of O₂ would have been extremely low since photosynthesis was just getting started.  Rubisco probably didn’t need to be too selective since O₂ was just a trace gas back then.

The selectivity of Rubisco for CO₂ over O₂ is affected by temperature.  Warmer temperatures decrease the selectivity making Rubisco even more inefficient.  That can be a problem for plants in a hot dry climate.  Also a change in the amounts of CO₂ to O₂ with respect to each other will influence the enzyme efficiency.  These two factors may become a significant concern in a world of global climate change where the both the temperature and concentration of CO₂ are on the increase.  How this could affect the world’s already insufficient food supplies will have to be seen.

Besides it’s affinity for reacting with a substrate, another characteristic of an enzyme is it’s rate of reaction called the Vmax.  Guess what, Rubisco’s Vmax also really sucks.  Probably not what you would expect for an enzyme that is the most abundant in the world.  Where as most enzymes catalyze thousands of reactions per second, Rubisco is only able to catalyze about 10 reactions per second.  Now, I hate to sound so judgmental, but that is really pathetic!  It is certainly possible that Rubisco was never able to evolve to be more efficient due to constraints on its structure once it became vital to the plant way of life.  Any alteration in the critical active site may have affected too many other protein-protein interactions necessary for normal function, and so never took place.  Alternatively, there may be some advantages to photorespiration, after all, so that completely shutting down that pathway would, likewise, be detrimental to growth.  There seems to be a trade off between having organisms who’s Rubisco has good affinity for CO₂ (K_m) and those who’s Rubisco has a fast reaction rate (Vmax).  It’s a case of, you can’t have your cake and eat it too.  If you favor one quality then you suffer in the other.

Plants have come up with a few smart ways of helping to boost the efficiency of Rubisco.  One way is to attempt to concentrate the amount of CO₂ around the enzyme.  C4 plants do this by adding the carbon from a CO₂ molecule to phosphenolpyruvate (PEP), then through a series of chemical reactions, the organic compound malate is produced.  The malate is shuttled to the plant cells that contain Rubisco and the CO₂ is removed.  This concentrates the CO₂ in the vicinity of Rubisco so it can act more efficiently.  The waste in energy to produce the malate is more than made up for by the better efficiency of the Rubisco in C4 plants due to this CO₂ concentrating ability.  C4 plants are a more recent evolutionary development, but only represents about 3% of land plants.  They are well suited for living in desert conditions where C3 plants would not be able to photosynthesize effectively and would rapidly lose too much water.  C3 plants to well in moderate climates with only moderate sun light.  The lower temperatures helps to improve Rubisco efficiency at utilizing CO₂ over O₂.  

 

C4 plants are therefore more efficient, especially in warm dry climates.  CAM (Crassulacean acid metabolism) plants close their stomata in the day to prevent fluid loss and open them at night to allow diffusion of CO2 into the leaves, where it is stored in malate.  During the day the CO₂ is again removed from the malate so it can be used by Rubisco to make carbohydrate.  CAM plants can be either land or aquatic.  

 

The C4 plant, Maize, busily concentrating CO2 to boost Rubisco efficiency

Point Three: Perhaps we can make a better Rubisco, one that can select CO₂  over O₂ more effectively, and react more quickly.   Nature has had billions of years to figure this out, so maybe its our turn now to design a Rubisco that can be improved in a variety of different ways.  This might be accomplished by artificial selection or genetic engineering – to produce a Genetically modified organism (GMO) with the desirable traits we choose.  In fact, there is a great deal of research looking into possible ways to improve Rubisco, but so far progress seems to have been rather modest.  A super Rubisco could in theory produce more carbohydrate under warmer, drier, and lower light conditions, decrease the amount of nutrient nitrogen necessary for plant growth, and remove more CO₂ from the atmosphere, and release more O₂.  This could be vital to consider for a growing global population that is outstripping its food resources and heading towards potential disaster due to global warming.  How could it be done?

It is known that red algae has a Rubisco with the highest value yet found for CO₂   affinity.  It is nearly 3 times better at discriminating CO₂ from O₂ than is the Rubisco from crops, like corn.  It may be theoretically possible to engineer crop plants to express the red algae Rubisco.   Other studies have looked to genetically engineering Rubisco by substituting key amino acid residues in critical areas of the enzymes protein structure and observing the effect.  This has resulted in some mild success.  In one study, by switching a particular alanine residue in the L subunit with a asparagine, the affinity was increased by 9%.  Not a huge increase, but potentially a good starting point.  

Other research has focused on speeding up Rubisco’s slow rate of reaction.  One way to accomplish this could be to create a CO₂ concentrating mechanism in C3 plants like corn and rice, that is similar to the natural CO₂ concentrating mechanisms found in C4 plants.  The ways to make this happen are less clear, but could involve manipulations that would put certain types of CO₂ transporter in the membranes of chloroplasts to help concentrate the gas where it needs to be.  

It should also be noted, that while the intended effects for changes to Rubisco protein would be for the common good of the planet, if we get to the point where such genetically modified plants are possible, it would need to be studied, not only to determine that there are no unintended consequences on the environment, but also that these changes actually result in greater plant growth and yield.  There may be some reasons why photorespiration is allowed to occur at the high rates it does.  One theory is that this is a protective mechanism for the plant so that in intense light conditions energy overload does not occur that could result in oxidative damage to the plant.  There may be a certain limiting factor where carboxylation can be maximized to a certain degree, but once you cross some threshold it actually becomes detrimental to the organism.  

There is no doubt that Rubisco is a curious and fascinating protein, and one on which our lives, and continued survival, are completely dependent.  It is certainly worthy of our admiration for its important and ancient role in maintaining earths biosphere.  There seems to be much more we need to understand about its biochemistry before we can tell if it will be a tool we can utilize to improve and protect our planet.  It could also potentially be altered in algae or cyanobacteria to terraform other planets like Mars, which although it has an extremely thin atmosphere, does have an abundance of CO₂ over O₂.  If we eventually discover life on other world that have evolved some form of photosynthesis, it will be interesting to learn what proteins or other methods they came up with to catalyze the carboxylation reaction that Rubisco serves for us here on earth.

References:

1. M. A. J. Parry, et al., “Manipulation of Rubisco: the amount, activity, function, and regulation”. Journal of Experimental Botany, Vol 54, No. 386,  pp. 1321-1333, May 2003.

2. Spencer M. Whitney, et. al., “Advancing our understanding and capacity to engineer natures CO2-sequestering enzyme, Rubisco”, Plant  Physiology, Vol. 155, pp. 27-35, Jan. 2011.

3. Wikipedia article on Rubisco:  https://en.wikipedia.org/wiki/RuBisCO

4. Wikipedia article on Photorespiration:  https://en.wikipedia.org/wiki/Photorespiration

5. Article on Mesign:     http://darwinskidneys.blogspot.com/2015/07/another-clever-mesign-brought-to-you-by.html

Fossil Friday: Thar She Blows; Whale evolution.

Thar She Blows: Evolution of whales!

by Rich Feldenberg

 

Welcome back to Fossil Friday.  The evolution of the cetacean group (marine mammals like whales and dolphins) is one of the coolest and most beautiful demonstrations of a clear link of fossil evidence from primitive forms to modern forms with many transitional fossils present.  

Whales, are of course, mammals and descended from air breathing land vertebrates.  All tetrapods descended from lobe finned fish (see my Tiktaalik post).  From there they diversified into amphibians, reptiles, mammals, and birds.  Some of the groups of reptiles (like the great marine reptiles during the age of the dinosaurs), and mammals (like the whales) returned to the sea many millions of years later.  Based on molecular genetics studies, the closest living land mammals to the whales is the hippopotamus.  

Cute little Pakicetus was one of the earliest known proto-whales.  These hoofed footed mammals were alive about 50 million years ago.  Based on bone structure of the skull around the auditory region, they fit into the cetacean group, but were not thought to be good swimmers.  Good swimmers in the family would come later!

It is thought that changes in the regulation of genes such as Sonic Hedge Hog (Shh) and Tbx4 may have been important in the loss of the hind limbs in the cetaceans.  By affecting when and how genes are expressed, major changes in structure can be made due to relatively small genetic changes. It is also pretty amazing to see the embryology of modern whales also betrays their ancestry. For example, in the whale fetus the nostrils start out in the usual position for a mammal, but as the maxillary bones grow to huge proportions this forces the nasal bones to the top of the skull. This type of evolutionary effect is called allometry and refers to a change in body parts due to changing the growth rate of different parts in relation to one another.

Over time the cetaceans evolved their characteristic echolocation apparatus, as well as, the development the blow hole from nostrils that were originally forward on the face.  Today, cetaceans are beautifully adapted for life in the oceans.

References and a cool video to watch:

1. Whale evolution Wikipedia:

https://en.wikipedia.org/wiki/Evolution_of_cetaceans

2. Animated video of whale evolution. This is pretty cool, check it out.

http://ocean.si.edu/ocean-videos/evolution-whales-animation

3. Sonic hedge hog:  Wikipedia

https://en.wikipedia.org/wiki/Sonic_hedgehog

4. Tbx4:   http://dev.biologists.org/content/130/12/e1205.full

Mutation Monday: OxoG is how radiation turns your own water against you!

by Rich Feldenberg

Welcome back to your mutation station.  Today we’ll examine how ionizing radiation breaks water molecules apart to form oxygen free radicles (or reactive oxygen species), which then go on to wreak havoc with your DNA.

Most of the damage done to us by ionizing radiation, such as X-rays and gamma rays, are not a consequence of direct hits to our DNA,  but are a secondary effect of the radiation splitting water into highly reactive and destructive molecules – the oxygen free radicles.  It is these oxygen free radicles that then go on to damage our cell’s vital components, like DNA.  Water is by far the most common molecule in our bodies, and statistically will be the most likely thing hit by an energetic photon of radiation that strikes us.

The oxygen free radicals are molecular species, such as the extremely reactive hydroxyl radical (*OH),  as well as hydrogen peroxide (H2O2) and the superoxide radical (*O2-).   These are often called oxygen free radicals, but not all of them are technically radicals (having an unpaired electron), so reactive oxygen species is really a more appropriate term.  These reactive molecules can then oxidize susceptible places on the DNA that lead to mutation.  Hydroxyl radical, is by far, the most reactive of the bunch, and basically reacts immediately with whatever is in it’s way as soon as it is formed.

1=singlet oxygen (higher energy state), 2=molecular oxygen, 3=superoxide radical, 4=hydrogen peroxide, 5=hydroxyl radical.

A common site of damage is the oxidation of the nucleotide base guanine (G) to produce 8-hydroxyguanine, also known as oxoG.  Whereas, normal guanine will base pair with cytosine (C), oxoG can base pair with both cytosine and adenine (A).  If oxoG happens to base pair with A, then after the next round of DNA duplication there will be a point mutation from the original G:C to the newly mutated T:A.  It turns out that this particular switch is very common in many tumor cells, and may be due to the damaging effects of radiation.

Oxo-G forming an inappropriate base pair with adenine

In this way, the effects of radiation are mainly by turning your own water against you.   In addition to radiation, oxygen free radicals are produced just by normal metabolism.  As we extract energy from sugar molecules, we pass electrons down the “respiratory chain – a set of enzymes in our mitochondria, that eventually react with Oxygen to form water.  During this process, free radicals are produced that have the same effect as those produced by water’s interaction with radiation.  It has been estimated that in just one year of breathing – something we all have to do if we are alive – is the equivalent of 10,000 chest X-rays worth of radiation.  Just being alive is dangerous!

References:

1. “Oxygen: the molecule that made the world”, by Nick Lane.  See chapter 6 (Treachery in the air) for some of the stats listed.   (a really great book, by the way).

2. “Molecular biology of the gene”, 7th edition, by James Watson;  ISBN-13: 978-0321762436 
Also an awesome text.